Considerable attention has focused on stem cells and their uses in a range of therapies. For example, the gap between the need for replacement of damaged or diseased organs in patients, with otherwise significant life-expectancy, and the supply of donor organs is growing at an ever increasing rate (Gridelli and Remuzzi, 2000). Tissue bioengineering and in vitro organogenesis research have the potential to bridge this gap. The availability of stem cells for organs in demand would greatly accelerate progress in these efforts. Age-dependent changes in stem cell function are predicted to contribute to the aging of human tissues (Merok and Sherley, 2001; Rambhatla et al., 2001). Stem cells are also ideal delivery vessels for gene therapy (Wilson, 1993; Brenner, 1996). In theory, after genetic engineering such stem cells would persist in a tissue while producing differentiated tissue constituent cells that would supply therapeutic gene expression.
The availability of liver stem cells that could give rise to mature functioning hepatocytes and other hepatic cells is particularly desirable. Hepatocyte transplantation has been proposed as a potential therapeutic method to treat irreversible liver failure and inherited hepatic disorders (Fujino et al., Cell Transplantation 10: 353-66 (2001)). In children, the most common indications of severe, irreversible liver disease are biliary atresia, a condition which leads to distortion of bile ducts and liver cirrhosis, and genetically transmitted metabolic disorders which may lead to hepatic failure and/or cirrhosis. Adults suffering from nonalcoholic or alcoholic cirrhosis as well as liver cancer are also transplantation candidates. Medical and surgical treatments often fail these patients.
One of the major limitations of hepatocyte transplantation therapy is the serious shortage of donor livers for hepatocyte isolation (Kobayashi et al., Cell Transplantation 10: 377-81 (2001)). An ideal alternative to primary human hepatocytes would be to use a clonal hepatocyte precursor cell line that grows in culture and retains the ability to express characteristics of differentiated, nontransformed hepatocytes following transplantation.
Such a precursor cell line has also been proposed as an attractive alternative to the use of primary human hepatocytes for developing a bioartificial, or hybrid artificial, liver (Kobayashi et al., Cell Transplantation 10: 377-81 (2001); Kobayashi et al., Cell Transplantation 10: 387-92 (2001)).
The liver is also a well-studied target organ for human gene therapy. For example, much attention has focused on the use of the liver as a target for gene therapy to treat diabetes. (Mulligan, Science 260:926-32 (1993); Crystal, Science 270: 404-410 (1995)).
Beyond their potential therapeutic applications, portable, stable cell lines that retain hepatocyte-specific metabolic activities would also be highly desirable to support development of human-specific drug metabolism assays.
Accordingly, methods to isolate and expand precursor stem cells, particularly without significant differentiation, are highly desirable. The availability of hepatocyte precursor stem cell lines would greatly contribute to cell replacement therapies such as liver cell transplants, gene therapies, tissue engineering, and in vitro organogenesis. Production of autologous stem cells to replace injured tissue would also reduce the need for immune suppression interventions. However, considerable difficulty in achieving this objective has been encountered thus far.
Cell growth is a carefully regulated process that responds to the specific needs of the body in different tissues and at different stages of development. In a young animal, cell multiplication exceeds cell loss and the animal increases in size; in an adult, the processes of cell division and cell loss are balanced to maintain a steady state. For some adult cell types, renewal is rapid: intestinal cells and certain white blood cells have a half-life of a few days before they die and are replaced. In contrast, the half-life of human red blood cells is approximately 100 days; healthy liver cells rarely die, and in adults, there is a slow loss of brain cells with little or no replacement.
Somatic stem cells possess the ability to renew adult tissues (Fuchs and Segre, 2000). The predominant way somatic stem cells divide is by asymmetric cell kinetics (see FIG. 1). During asymmetric kinetics, one daughter cell divides with the same kinetics as its stem cell parent, while the second daughter gives rise to a differentiating non-dividing cell lineage. The second daughter may differentiate immediately; or, depending on the tissue, it may undergo a finite number of successive symmetric divisions to give rise to a larger pool of differentiating cells.
Attempts at deriving hepatocyte precursor stem cell isolation have been described, for example, in studies to enrich for hematopoietic stem cells (Phillips et al., 2000). However, although high degrees of enrichment have been reported, so far somatic stem cells, including hepatocyte precursor stem cells, have neither been identified nor purified to homogeneity. A major obstacle to these two challenges is the inability to expand HSCs in culture.
Attempts at propagating somatic stem cells have encountered a number of significant difficulties. The asymmetric cell kinetics which are a defining characteristic of somatic stem cells are also a major obstacle to their expansion in vitro (FIG. 1) (Merok and Sherley, 2001; Rambhatla et al., 2001). In culture, continued asymmetric cell kinetics results in dilution and loss of an initial relatively fixed number of stem cells by the accumulation of much greater numbers of their terminally differentiating progeny. If a sample includes both exponentially growing cells as well as somatic stem cells, the growth of the exponentially growing cells will rapidly overwhelm the somatic stem cells, leading to their dilution.
Even in instances where it is possible to select for relatively purer populations such as hematopoietic stem cells (for example by cell sorting), these populations do not expand when cultured.
The liver contains several resident cell types in addition to hepatocytes, including stellate cells, cholangiocytes, oval cells, Kupffer cells, and sinusoidal endothelial cells (Alpini et al., 1994). In the adult liver, the majority of liver cells are hepatocytes, with stellate cells and cholangiocytes representing minority populations of cells (Grompe et al., 2001). Stellate cells function as the primary source of extracellular matrix in normal and diseased liver, transitioning from a quiescent vitamin-A rich cell to a highly fibrogenic cell during activation caused by liver injury. Cholangiocytes line the intrahepatic biliary tree inside the liver. Cholangiocytes play a key role in the modification of bile, secreted by hepatocytes, by a series of reabsorbtive and secretory processes under both spontaneous and hormone-regulated conditions. Cholangiocytes also have the ability to selectively proliferate during injury such as bile duct ligation. Oval cells are found in the periportal region of the liver under some conditions, and have been postulated to function as a bi-potential precursor cell with the ability to give rise to hepatocytes and cholangiocytes (also known as bile duct cells).
The possible existence of renewing stem cells in adult liver has been hotly contested for many years. Because mature hepatocytes divide in response to liver injury, many have considered their division to provide the renewing stem cell function for adult liver. Although a well-defined hepatocyte stem cell turnover unit has been elusive, it is clear that both cell division and apoptosis do occur in adult liver.
Recent studies in rodents indicate that hepatocyte stem cells (derived from the mesoderm) may be able to home to the liver after it is damaged, and demonstrate plasticity in becoming hepatocytes (usually derived from the endoderm). Lagasse et al., Nat. Med. 6:1229-34 (2000); Petersen et al., Science 284: 1168-70 (1999); Theise et al., Hepatology 32: 11-16 (2000). However, there is no clear indication that cells from the bone marrow normally generate hepatocytes in vivo. Moreover, it is not known whether this kind of plasticity occurs without severe damage to the liver or whether hepatocyte stem cells from the bone marrow generate oval cells of the liver. Crosby et al., Gut 48:153-4 (2001). Although hepatic oval cells exist in injured livers, it is not clear whether they actually generate new hepatocytes. Indeed, hepatocytes themselves may be responsible for the well-known regenerative capacity of the liver.
The possible existence of a “transitional stem cell” in liver that is capable of giving rise to both hepatocyte and bile epithelial precursor cells has also been suggested. Although bi-potent progenitor cells of this type have been described during embryonic liver development (Zaret, Ann. Rev. Physiol. 58: 231-251 (1996); Sell, Modern. Pathol. 7: 105-112 (1994)), there is no clear indication that this cell exists in the adult liver. Oval cells are found in the periportal region of the liver under some conditions and may represent such a bi-potent progenitor cell.
Previously, rat liver-derived cell lines have been described from three different liver states, fetal liver, pathological liver (i.e., oval cells), and normal adult liver. The best described of these is the clonally-derived line WB-F344, which was isolated from an adult rat (Tsao et al., Exp. Cell Res. 154: 38-52 (1984)). WB-F344 cells have many properties indicative of hepatocyte stem cell origin and have been cited as the best evidence for the existence liver stem cells. These cells express several markers of hepatocyte and biliary origin. When transplanted in vivo, WB-F344 cells have been shown to integrate into hepatic plates and express hepatocyte specific marker proteins. However, WB-F344 cells phenotypically most closely resemble oval cells and do not represent a true hepatocyte precursor cell line. For example, they express oval cell-specific antigens, like OV6, which are not expressed by mature hepatocytes (Grisham et al., P.S.E.B.M. 204: 270-279 (1993); Grompe et al., “Liver Stem Cells” in Stem Cell Biology, Marshak, Gardner, and Gottleib, eds. CSHL Press (Cold Spring Harbor) pp. 455-497 (2001)).
Accordingly, it would be particularly desirable to have a source of hepatocyte precursor stem cells, including hepatocyte progenitor cells which can be expanded in vitro and express hepatocyte-specific characteristics, including metabolic activities, as well as cholangiocyte progenitor cells which can be expanded in vitro and express cholangiocyte-specific characteristics.